In order to discuss the nonylphenol (NP) biodegradability effect in the wastewater treatment process, the primary metabolites and route of NP had been analyzed in the molecular level by the interpretation of gas chromatography mass spectrometry (GC-MS) and the two experimental methods. A method was that activated sludge degraded two types of simulating sewage comparatively. Type-1 wastewater was made up of water, glucose, peptone and inorganic salts, and type-2 sewage was mixed up of NP and type-1 as comparison wastewater. The other experiment was that dominant bacteria group metabolized the culture medium contained NP as only organic carbon source. The results show that the NP primary metabolites from the activated sludge experiments are a series of short chain alkyl phenols with butyl, amyl or hexyls. There are different methyl branch structures correlated with isomerous nonyl of NP in the alkyls. The primary metabolites from the dominant bacteria experiment have alkyl phenols and benzene acetic. The bio-degradation pathways of NP would be that the long branches in nonyl of NP are firstly degraded into short methyl branches, which come into being a series of short chain alkyl phenols with different branch structures. The middle products should be further metabolized into simpler compounds, such as benzene acetic. Based on the endocrine disrupter characteristic of alkyl phenol compounds, the short chain alkyl phenol compounds still have estrogen hormone effect, and their environmental effect must be pay attention in the pollutant control of NP.